High-resolution microscopy methods are particularly important in the field of microscopy at present. These are microscopy methods which achieve a local resolution in a sample that is increased beyond the optical resolution limit which results according to Abbe's theory. Such microscopy methods are e.g. PALM, STORM, d-STORM or GSDIM. They are based on the highly precise localization of individual fluorescent fluorophores in that it is ensured that the fluorophores fluoresce in as isolated a manner as possible. It is then possible, for the recorded radiation of such an isolated fluorophore, to determine the location of the fluorophore with a spacial resolution that exceeds the diffraction limitation, thus Abbe's theory. The location is determined with a precision up to the nanometer range using highly sensitive cameras in widefield. If this process is repeated several times for the sample, such that as far as possible all fluorophores have been imaged, isolated, once and localized, an image can be assembled from the several frames.
Localization-based high-resolution microscopy thus images a sample in different light states and achieves a local resolution in the subwavelength range of the visible spectral range, i.e. of light.
Tests that show the resolution are indispensable for the development of such microscopy methods and microscopes, but also for testing existing systems, for fault finding and, not least, for demonstrating and marketing high-resolution microscopes. For this, samples are needed, the structures of which are well-known, in order to test whether the microscope can image these known structures with the desired resolution.
Although it is known in the state of the art to produce periodic structures with defined sizes or spacings and to use these as test samples for microscopy, these test samples are not suitable for the mentioned localization-based high resolution approach. For the reasons mentioned at the beginning, localization-based high-resolution microscopy requires fluorescence molecules which can be excited to fluorescence radiation individually. Periodic structures with defined sizes and spacings do not meet this requirement.
Furthermore, resolution tests are known in the state of the art which modulate the amplitude or phase of the illuminating light. Such resolution tests are also not suitable for localization-based high-resolution microscopy, thus for a microscope which images a sample in different light states.
Therefore, biological samples that have correspondingly marked structures are currently used for these microscopy methods. The following disadvantages result here:
1. The samples have a low durability. Therefore, it is not possible to prepare these samples beforehand and send them to a user.
2. In the case of biological samples, reproducibility is limited in principle. Thus it is not known precisely which structure is present in the test.
3. Biological samples are complex to handle, they require for example corresponding culture media, buffers etc., which precludes a simple checking or demonstration of a localization-based high-resolution microscope.
4. Finally, the structures used are not strictly predefined, as biological samples always have a certain variability. A resolution test that can be repeated is thus unachievable.
In the state of the art, the use of a so-called DNA origami was proposed by Steinhauer et al., Angew. Chem., 121, 2, 2009. Fluorophores are bound at particular points in such DNA origami structures, with the result that two fluorophores are arranged with a particular spacing in the sub-100 nm range. However, the named limitations with respect to durability and handling also exist in these samples, as the fluorophores first have to be brought into their switchable state by a chemical redox system. In addition, the spacings of the fluorophores are not as well-defined as is desired, because DNA structures bend. It is also difficult to achieve the binding of the DNA structures to a substrate surface in such a defined way that there are no differences between the theoretically expected spacing of the bound fluorophores and the real spacing influenced by projection effects. In addition, it is possible to attach only one or a few molecules per binding position. As fluorophores normally bleach in localization-based high-resolution microscopy, the samples would, as a result, only be usable for quite a short time. In addition, there are localization microscopes which expect a particular blinking statistic of the fluorophores or require a modification of this blinking statistic. This is also not possible with the DNA origami structures according to Steinhauer.